Chemotherapeutic Agents Impair ARHGAP29/IRF6 Expression in Oral Mucositis Cell Models

Objectives: Cancer therapy-induced oral mucositis (CTOM) is a toxic side effect of chemotherapy resulting in ulceration of the oral mucosa which may disrupt cancer treatment. A single nucleotide polymorphism (SNP rs4847278) within an enhancer region of ARHGAP29 was previously found to be associated with CTOM. ARHGAP29, essential to the integrity of the oral mucosa is positively regulated by IRF6, a mediator in epithelial barrier defense. Cisplatin and Doxorubicin are commonly used chemotherapeutic agents contributing to CTOM development. Our objective was to determine the effects of cisplatin and doxorubicin in cell culture models for CTOM.
Methods: Cell cultures were dosed with various concentrations of Cisplatin and Doxorubicin for 24 hours. Cytotoxicity assays (n=9) were performed using oral keratinocyte cell line OKF6-TERT2, buccal mucosa cell line HO-1-N-1, and vascular endothelial cell line HUVEC-TERT2. Immunofluorescent images (n=3) were taken in Cellsens Standard software to determine IRF6 and ARHGAP29 expression in each cell line. Statistical analysis was performed in GraphPad-Prism using Mann-Whitney U test (α=0.05).
Results: HUVEC cells showed increased cell viability 24h-post dosing with cisplatin at 0.1µM/1µM treatment levels (p<0.0001), with OKF6 cells displaying a similar trend. OKF6 and HO-1-N-1 cell viability was significantly reduced at 10µM concentration (p<0.0001), while HUVEC cell viability was increased (p<0.0001). At time point 48hrs OKF6 showed decreased viability at 1µM (p<0.0001). Doxorubicin treatment resulted in steadier decrease in viability in each cell line. At 48hrs OKF6 viability decreased at 1µM (p<0.0001) while HO-1-N-1 and HUVEC steadily decreased from 1-10µM (p<0.05). Immunofluorescence microscopy showed that both drugs affected IRF6 and ARHGAP29 expression in a cell-specific manner.
Conclusions: The overall effects of doxorubicin and cisplatin treatments on IRF6 and ARHGAP29 expression were cell-specific. These agents initially induced transition of the cells to an accelerated healing/ growth state at lower doses, while harming the cells significantly at longer exposures.